In the world of micro arrays or biochips, biological molecules (e.g., oligonucleotides, polypeptides, oligopeptides and the like) are placed onto surfaces at defined locations for potential binding with target samples of nucleotides or receptors or other molecules. Microarrays are miniaturized arrays of biomolecules available or being developed on a variety of platforms. Much of the initial focus for these micro arrays have been in genomics with an emphasis of single nucleotide polymorphisms (SNPs) and genomic DNA detection/validation, functional genomics and proteomics (Wilgenbus and Lichter, 1. Mol. Med. 77:761, 1999; Ashfari et al., Cancer Res. 59:4759, 1999; Kurian et al., J. Pathol. 187:267, 1999; Hacia, Nature Genetics 21 suppl.:42, 1999; Hacia et al., Mol. Psychiatry 3:483, 1998; and Johnson, Curr. Biol. 26: RI71, 1998).
There are, in general, three categories of micro arrays (also called “biochips” and “DNA Arrays” and “Gene Chips” but this descriptive name has been attempted to be a trademark) having oligonucleotide content. Most often, the oligonucleotide micro arrays have a solid surface, usually silicon-based and most often a glass microscopic slide. Oligonucleotide micro arrays are often made by different techniques, including (1) “spotting” by depositing single nucleotides for in situ synthesis or completed oligonucleotides by physical means (ink jet printing and the like), (2) photolithographic techniques for in situ oligonucleotide synthesis (see, for example, Fodor U.S. patent '934 and the additional patents that claim priority from this priority document, (3) electrochemical in situ synthesis based upon pH based removal of blocking chemical functional groups (see, for example, Montgomery U.S. Pat. No. 6,093,302 the disclosure of which is incorporated by reference herein and Southern U.S. Pat. No. 5,667,667), and (4) electric field attraction/repulsion of fully-formed oligonucleotides (see, for example, Hollis et al., U.S. Pat. No. 35 5,653,939 and its duplicate Heller U.S. Pat. No. 5,929,208). Only the first three basic techniques can form oligonucleotides in situ that are, building each oligonucleotide, nucleotide-by-nucleotide, on the micro array surface without placing or attracting fully formed oligonucleotides.
With regard to placing fully-formed oligonucleotides at specific locations, various microspotting techniques using computer-controlled plotters or even ink-jet printers have been developed to spot oligonucleotides at defined locations. One technique loads glass fibers having multiple capillaries drilled through then with different oligonucleotides loaded into each capillary tube. Microarray chips, often simply glass microscope slides, are then stamped out much like a rubber stamp on each sheet of paper of glass slide. It is also possible to use “spotting” techniques\ to build oligonucleotides in situ. Essentially, this involves “spotting” relevant single nucleotides at the exact location or region on a slide (preferably a glass slide) where a particular sequence of oligonucleotide is to be built. Therefore, irrespective of whether or not fully-formed oligonucleotides or single nucleotides are added for in situ synthesis, spotting techniques involve the precise placement of materials at specific sites or regions using automated techniques.
Another technique involves a photolithography process involving photomasks to build oligonucleotides in situ, base-by-base, by providing a series of precise photomasks coordinated with single nucleotide bases having light-cleavable blocking groups. This technique is described in Fodor et al. U.S. Pat. No. 5,445,934 and it's various progeny patents. Essentially, this technique provides for “solid-phase chemistry, photolabile protecting groups, and photolithography . . . to achieve light-directed spatially-addressable parallel chemical synthesis.” Binary masks are used in the preferred embodiment.
The electrochemistry platform (Montgomery U.S. Pat. No. 6,093,302, the disclosure of which is incorporated by reference herein) provides a micro array based upon a semiconductor chip platform having a plurality of micro electrodes. This chip design uses Complimentary Metal Oxide Semiconductor (CMOS) technology to create high-density arrays of microelectrodes with parallel addressing for selecting and controlling individual micro electrodes within the array. The electrodes turned on with current flow generate electrochemical reagents (particularly acidic protons) to alter the pH in a small defined “virtual flask” region or volume adjacent to the electrode. The micro array is coated with a porous matrix for a reaction layer material. Thickness and porosity of the material is carefully controlled and biomolecules are synthesized within volumes of the porous matrix whose pH has been altered through controlled diffusion of protons generated electrochemically and whose diffusion is limited by diffusion coefficients and the buffering capacities of solutions.
The micro array systems have detection processes generally using some form of photon based detection. That is, most detection processes use fluorescent probes (alternatively visible dyes or luminescent probes) attached to “target” DNA sequences to detect binding or hybridization to an oligonucleotide capture probe attached on a micro array. Depending upon the intensity of the signal, such micro arrays having probes to show hybridization have to be read through laser confocal microscope-based system for micro arrays configured in a monolayer (such as those micro arrays made through high density spotting or photolithography techniques) or by a video-type camera (such as a CCD camera) for those micro arrays having a three-dimensional matrix for each spot in high density formats. In each instance, there is often stray light or other noise signals that cause false readings to be made. Moreover, it occasionally becomes difficult to distinguish between shades of gray or barely perceptible signals as true positives or false positives. Therefore, there is a need in the art for improvements to the detection/reading process for analyzing microarrays. The present invention was made to address this need and to provide a detection system that can generate a more objective “yes” or “no” answer for each site in high density micro array detection.